Transfer method for specific cellular localization of nucleic acids

ABSTRACT

The present invention relates to a novel method of genetic modification, wherein a nucleic acid of interest is transferred across a biological membrane, and/or directed to a specific location within or on a cell, by use of a synthetic transport entity. The transport entity according to the invention is new as such and produced by coupling a functional element (FE), such as a nuclear localization signal (NLS), an antennapedia peptide of a protein comprising both membrane translocation and nuclear transport properties, to a binding element (BE), such as a peptide nucleic acid (PNA), preferably separated by a linker molecule, which combination is then hybridized to a BE target sequence present on a carrier, which also includes the nucleic acid of interest. The present nucleic acid of interest may for example be a gene encoding a peptide, a protein or an RNA, or any other nucleic acid useful in genetic recombination events.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/SE99/00398 which has an Internationalfiling date of Mar. 15, 1999, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a method for transferring a nucleicacid, a derivative or an analogue thereof across a biological membrane,and/or directing it to a specific location within or on a cell, by useof a novel synthetic transport entity.

BACKGROUND

Methods for genetic modification, wherein exogenous genetic material isintroduced into host cells to provide a function thereof, are usuallylimited by the rate of the uptake of the genetic material introducedinto the cells. In eucaryotic cells, the nuclear uptake is oftenlimiting. Even though direct injection methods have been used in thiscontext, they are, however, extremely slow and labor-intensive. Thus,for use in larger scales, standard methods for transferring nucleicacids into cells are rather based on an uptake of complexes formedbetween different chemical compounds of nucleic acids. The geneticmaterial is then left to enter the nuclei of the cells passively.

Nuclear localization signals (NLS) have been proposed in this context.As one example, Sebestyen et al. (Nat. Biotechnol, 1998, January;16:(1):80-85) have suggested to use digitonin permeabilized cells toenable nuclear translocation after chemically linking an NLS peptide toa plasmid.

Further, Yoneda et al. (Exp. Cell. Res. 201:213 (1992)) have reportedtranslocation of proteins larger than 970 kDa into the nucleus. Morespecifically, a fusion protein containing a nuclear localization signal(NLS) is transported into the nucleus of a cell.

U.S. Pat. No. 5,539,082, in the name of Nielsen et al, discloses a classof compounds known as peptide nucleic acids (PNAs). The PNAs describedtherein may comprise ligands, such as DNA bases, conjugated to a peptidebackbone through a suitable linker. The PNAs according to U.S. Pat. No.5,539,082 may e.g. be exploited to target specific genes and viruseswithin a cell, while the properties thereof are characterised by anabsence of charge and a water solubility.

Further, WO 96/11205, also in the name of Nielsen et al, proposes a PNAconjugate comprised of a PNA chemically bound to a conjugate, such asany one of a large number of molecules, all of which are aimed atproviding the PNA with desired properties. Accordingly, the PNA isuseful as such, for example as a diagnostic or therapeutic agent. SaidPNA is, similar to the above discussed U.S. Pat. No. 5,539,082, intendedfor exerting its advantageous functions within a cell.

SUMMARY OF THE PRESENT INVENTION

The object of the present invention is to provide a general andefficient method of genetic modification, wherein a nucleic acid ofinterest, a derivative or an analogue thereof is transferred across abiological membrane, and/or directed to a specific location within or ona cell, by a novel synthetic transport entity. The present transportentity is according to the invention provided by coupling a functionalentity (FE), which may represent any kind of desired biologicalproperty, to a binding element (BE), such as a peptide nucleic acid(PNA), optionally with a linker molecule for keeping said FE and BEapart; and hybridisation thereof to a BE target present on a carrier ofthe nucleic acid of interest. The invention also relates to the noveltransport entity as such as well as to various advantageous usesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing the target site in theanti-sense Cy-5 oligonucleotide hydridising to a sense PNA-NLS dualfunction peptide according to the invention.

FIG. 2 illustrates nuclear translocation of fluorescence labelledoligonucleotides.

FIG. 3 shows nuclear, cytoplasmic and Golgi-like ratio of Cy-5/Cy-3fluorescence.

FIG. 4 is a shift assay of antisense and sense oligonucleotides withPNA-NLS dual function peptide according to the invention.

FIG. 5 illustrates the effect of a transport entity according to theinvention: The effect of PNA-NLS when hybridised to PNA targetcontaining EGFP plasmids and the effect of removing the excess PNA-NLSmolecules from the transfection mix are shown.

FIG. 6 shows lacZ and EGFP transfections with or without the addition ofPNA-NLS and after purification of excess PNA-NLS.

DEFINITIONS

In the present specification, the following terms and abbreviations areused as follows:

As used herein, the term “a nucleic acid of interest” relates to anyDNA, RNA or other nucleotide, or any analogue or derivative thereof,useful to perform a genetic modification of a cell. In other words, itmay be desired either to transfer such a “nucleic acid of interest”across a biological membrane, into a cell or a cell nucleus, and/or todirect said “nucleic acid of interest” to a specific location in such anew environment. A “nucleic acid of interest” may for example be a geneencoding a peptide or a protein, such as en enzyme, provide a regulatingfunction, such as a binding site, etc.

The term “functional element” (FE) relates to any moiety capable ofconferring one or more specific biological functions or properties to amolecule linked to it. It may e.g. provide a transporting capability.The present functional elements will be further examplified in thefollowing detailed description of the invention.

A “binding element” (BE) may be any natural or synthetic nucleic acid,nucleic acid derivative or nucleic acid analogue capable of specific,strong and durable binding to a specified target thereof, preferably byhybridisation. One example of such a BE is the PNA described below.

Thus, a “PNA” refers to a Peptide Nucleic Acid and more specifically aDNA mimic with a pseudopeptide backbone consisting of aminoethyl glycineunits, to which the nucleobases are attached via methylene carbonyllinkers. (See e.g. Nielsen, P. E. Peptide nucleic acid (PNA): “A leadfor gene therapeutic drugs”, Perspectives in Drug Discovery and Design,Vol. 4, pp. 76-84; and Dueholm of al., New J. Chem., 1997, 21, 19-31:“Chemistry, properties and applications of PNA”.) A PNA molecule iscapable of hybridising to complementary ssDNA, dsDNA, RNA and PNAtargets. In the present patent application, it is to be understood thatthe term “PNA” refers to any DNA analogue comprised of the abovementioned backbone and nucleobases, and the term should thus not belimited to the specific structures disclosed in the reference givenherein.

“NLS” refers to a nuclear localization signal, which may be anyprotein/peptide that recognizes and binds specifically to residues oncertain transport proteins. More specifically, NLS domains are aminoacid sequences which have evolved in poly-peptides, thereby facilitatingmigration of a polypeptide from the cytoplasm into the nucleus.Specified nuclear polypeptides containing NLS domains have been shown toenable the transport of a polypeptide-RNA complex into the nucleus(Mattaj and DeRobertis, 1985).

The term “vector” is used herein to denote a plasmid, anoligonucleotide, or any other molecule or construct capable ofharbouring and/or transferring nucleic acids during genetic modificationevents. Thus, the term “vector” will also relate to any analogue orderivative of the ones examplified above.

The term “cell wall” as used herein relates to any membrane that servesto surround a living organism, such as a eucaryotic cell membrane, themembrane surrounding a plant cell or a bacterium etc.

“Transfection” is used herein as a general term for any uptake by a cellof genetic material from the culture medium.

In the present context, a “transforming sequence” relates to anysequence that participates in a genetic modification event in a hostcell, and may e.g. be a protein coding sequence, regulatory elements, anentire gene etc.

The term “recombinant” when referring to a cell is used herein simply todenote that a genetic modification has occurred therein. Morespecifically, it is used to indicate that a modification thereof hasbeen obtained by the introduction of an exogenous nucleic acid, by thealteration of a native nucleic acid or that the cell is derived from acell so modified.

The term “cell” or “host cell” is used herein to denote any cell,wherein any foreign or exogenous genetic material has been introduced.In its broadest sense, “host cell” is used to denote a cell which hasbeen genetically manipulated.

The term “polymer”, such as a “protein”, “polypeptide” and “peptide”,are used interchangeable herein and relates to any shorter or longerpolymer of amino acid residues. In addition to naturally occurring aminoacid polymers, the term also applies to amino acid polymers, wherein oneor more amino acid residues are artificial chemical analogues of thecorresponding naturally occurring amino acids. Further, the term“polymers” in the present context is also understood to include anyglycoproteins, lipids, lipoproteins etc. useful for the herein disclosedpurpose.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunological or chemical means.

The term “hybridise” refers herein to any binding, duplexing orhybridisation by base pairing of complementary bases of nucleic acids orpeptide nucleic acids, or any derivatives or anaologues thereof.

The term “specific hybridisation” as used herein refers to the binding,duplexing or hybridisation of a molecule only to a particular nucleotidesequence when that sequence is present in a complex mixture or DNAand/or RNA, such as in a cellular environment.

As used herein, “homologous” sequences are sequences, which areidentical or sufficiently similar to cellular DNA such that thetargeting sequence and cellular DNA can undergo specific base pairing.

As used herein, a “targeting sequence” is a sequence, which directs thenucleic acid of interest to a desired site in a genomic orextra-chromosomal DNA or RNA-sequences contained in a target cell.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a method oftransferring a nucleic acid of interest across a biological membraneand/or directing such a nucleic acid to a specific location within or ona cell by use of a synthetic transport entity; which comprises the stepsof

(a) providing a carrier molecule comprising the nucleic acid of interestand a binding element (BE) target sequence, optionally as one element;

(b) providing a complex by coupling at least one functional element (FE)to a BE;

(c) hybridising the BE of said complex to the BE target of said carrier;and

(d) contacting said transport entity with said biological membrane toprovide for a transfer of the nucleic acid of interest across saidmembrane.

In a preferred embodiment of the present method, the binding element(BE) is a peptide nucleic acid (PNA) or a derivative or an analoguethereof capable of a specific and durable hybridisation to a PNA targetsequence. The biological membrane may, for example, be a cell wall or anuclear membrane. The complex created in step (b) may also comprise aspacer element, such as a linker (L) molecule, which separates the BEfrom the FE. In specific embodiments discussed in more detail below, thecomplex is comprised of more than one FE, in which case more than onelinker may be used for separation. Step (a) may also include theinsertion of a detectable marker or label in the carrier, which willfacilitate a subsequent analysis of the efficiency of a geneticmodification performed by this method.

Thus, in the present method, the advantageous properties of a bindingelement (BE), preferably a PNA or a PNA-like molecule, are primarilyused in a first step of genetic transformation, preceding the actualtransfer of a nucleic acid of interest into a cell or a nucleus, inwhich the effect of said nucleic acid of interest is desired. Thus,contrary to the prior art patents discussed in the introduction of thepresent specification, the advantage of the method according to theinvention is not based, or relying, on the PNA's or binding element'scapability to enter a cell or a nucleus. Rather, a PNA, or anyequivalent binding element, is used according to the invention toprovide a strong and satisfactorily coupling of an functional element(FE), via the PNA/PNA target interaction, to a carrier of a nucleic acidof interest. Thus, the FE will be used according to the invention toprovide for the actual transport of the whole transfer entity across thebiological membrane.

The PNA used in the preferred embodiment of the present method is asynthetic DNA analogue, that binds strongly to DNA and RNA with a higheraffinity than DNA-DNA, RNA-DNA or RNA-RNA binding. The specific sequencethereof is designed in order to be specific for the nucleic acid towhich it is intended to bind by hybridisation. PNA is metabolized veryslowly and has also been shown to be non-toxic, which evidently is agreat advantage when used in the pharmaceutical field. In addition, PNAis capable of a highly specific binding to the sequence of the nucleicacid that is complementary thereto, which in turn will provide a highfrequency of correctly transformed cells when a PNA is used as BE in thepresent method for genetic transformation. Thus, the use of PNA as BE inthe present method will confer excellent RNA and DNA hybridisationproperties and biological stability to the complex formed. PNA is easilyproduced by someone skilled in this field by solid phase peptidesynthesis (see e.g the international patent applications WO 95/01370 andWO 92/2072 and Nielsen, P. E.: Peptide nucleic acid (PNA): “A lead forgene therapeutic drugs”, Perspectives in Drug Discovery and Design, Vol.4, pp. 76-84).

In a preferred embodiment, the above mentioned linker sequence isessentially or completely uncharged. In this context, the term“non-reactive” is used to explain that the linker will not undergo anyundesired or deleterious chemical reactions in the environment where itis used, e.g. that it does not react with any other components of thecell and/or nucleus that it contacts during a transfection process. Itshould also be essentially non-reactive as regards any reagents used inthe present method as well as in view of the BE, such as PNA, and thefunctional element (FE), carrier etc. The present linker may e.g. becomprised of a polymer of a suitable number of amino acid residues, eventhough it is to be understood that any other molecule which functions asa spacer element without interfering with the desired result may beused. The size and nature of the linker sequence is dependent on thesurrounding elements, as the primary function thereof is to provide asufficient spacing between said elements to enable the effect of thedesired function. Further, it should not interfere with the desiredeffect or expression of the nucleic acid of interest after transferacross the membrane.

In a specific embodiment, the present linker is cleavable by enzymes,such as cellular proteases or nucleases, enabling a transport across abiological membrane and/or adherence to a cell surface receptor, andthen a subsequent disposal of the part or parts which are not desirablewithin the target, such as within the cytoplasm or the nucleus. In oneparticular embodiment, the BE is cleaved off, in order to provide for amore efficient effect of the inserted nucleic acid of interest. Inanother exemplary embodiment of the present method, the linker iscapable of essentially hiding or masking the present transport entity,e.g. from undesired biological degradation, such as by proteases. Inaddition or alternatively, the linker may possess any furtheradvantageous property, such as conferring another biological functionalin itself.

The present functional elements (FEs) may provide any number offunctions, such as a structural function, e.g. binding to a cellmembrane target molecule, or an enzymatic function, e.g. an integraseactivity, to enhance site-specific insertion of transferred DNA.Specific examples of advantageous FEs suitable for use according to thepresent invention are e.g. the function of cellular attachment, forexample via transferrin receptors; cell membrane penetration, forexample via antennapedia peptides; a nuclear transport, which will bediscussed in more detail below; nucleic acid condensation peptides,preferably with strong, positive charge; endosome/lysosome escape, forexample via adenovirus capsid proteins; and DNA integration, e.g. via anintegrase. In one particular embodiment, the FE according to theinvention is a protein of the HIV virus denoted TAT. In a anotherspecific embodiment of the present method, the FE is an endosomedisrupting component, which prevents degradation of the transferredbiological element by the cellular process of lysosomal degradation.

In one embodiment of the present method, the biological membrane to bepenetrated is the membrane or wall surrounding a eucaryotic orprocaryotic cell. In an additional or alternative embodiment, thepresent method is used to insert a nucleic acid of interest into thenucleus of a eucaryotic cell. In the last mentioned embodiment, anefficient transport may be provided by creating a complex of a BE, suchas a PNA, and a suitable nuclear localization signal (NLS) as the FE, oras a part of the FE.

In the present methods, a transport entity constituted of a BE and morethan one of the mentioned FEs and linkers, in a suitable order,depending on the intended use and the desired result, may be used. Thus,as one example, a sequence of BE-linker-FE-linker-FE etc. may be used.Further, in a specific embodiment, the carrier will comprise more thanone BE target sequence, thus enabling the hybridisation of more than oneof the various BE-linker-FE complexes thereon. In this specificembodiment, there is no requirement that the BEs and/or FEs areidentical. On the contrary, it may be advantageous to include differentones. Thus, in a specific embodiment, the complex may contain aPNA-linker-FE-linker-FE-sequence. The linkers are made to providesuitable spacings, depending e.g. on sizes and other properties of theelements. Further, the present carrier may include more than one targetfor a BE, such as PNA. Accordingly, such a carrier is capable ofhybridisation to more than one of the herein examplified complexes.

Further, the present invention relates to a method as disclosed aboveused for a diagnostic purpose. Thus, the nucleic acid of interest mayencode a diagnostic marker or label and the method may be exerted invivo, in order to enable a subsequent diagnosis of a subject in needthereof.

In addition, the present invention also relates to a kit suitable forperforming any one of the methods disclosed herein. Such a kit maycontain a binding element (BE), such as a PNA, or functional fragmentsthereof; a functional element (FE); such as a nuclear localizationsignal (NLS) or antennapedia peptide; a double-stranded oligonucleotidecomprising target sites for said BE, such as a PNA target sequence. Inone embodiment of the present kit, the FE enables the transfer and/ordirection of the transport entity. In an alternative embodiment, the kitaccording to the invention also comprises suitable reagents for suchtransfer and/or direction. The kit according to the invention ispresented in a suitable container, optionally containing instructions tofacilitate the use thereof in appropriate methods.

In a second aspect, the present invention relates to a novel synthetictransport entity as such, which transport entity is useful in any one ofthe above discussed methods. The present BE-FE complex may be describedby the general formula I:

. . . BE-L-FE . . .   (I),

wherein

BE denotes a binding element;

L denotes a linker, which however is an optional element; and

FE denotes a functional element.

Said complex is then capable of a sequence specific hybridisation to aBE target present on a carrier, which in addition to, or as a part of,the present BE, contains one or more nucleic acids of interest. Thecarrier may e.g. include a plasmid or a functional part thereof, such asa gene, an oligonucleotide, or a chimeraplast (see e.g. Cole Strauss,A., el al., in Science, vol 273, September 1996: “Correction of theMutation Responsible for Sickle Cell Anemia by an RNA-DNAOligonucleotide”). The BE may as mentioned above preferably be a PNA.The use of a linker according to the invention is optional, butpreferred and it is described in detail above in relation to the firstaspect of the invention.

Accordingly, one embodiment of this second aspect is a synthetictransport entity suitable for transporting a nucleic acid of interestacross a biological membrane, such as a cell wall, and/or directing saidnucleic acid to a specific location within, or on, a cell.

Further, in this second aspect of the invention, it is to be understoodthat the transport entity may include more than one of the mentioned FEsand linkers, in any suitable order, depending on the intended use andthe desired result. Thus, in a specific embodiment, the complex maycontain a BE-linker-FE-linker-FE-sequence. The carrier used ispreferably a plasmid or an oligonucleotide which comprises one or moreBE targets as well as one or more nucleic acids of interest. The strongsequence specific hybridisation of the BE/BE target will thus attach thecomplex to the carrier and the FE will preferably provide a transferacross the membrane in question. Linker molecules are included asappropriate.

Further, in another embodiment of this second aspect, the presentinvention relates to a transport entity especially suitable fortransporting one or more nucleic acids of interest across a nuclearmembrane of a eucaryotic cell, which is comprised of a BE, preferably apeptide nucleic acid (PNA), complexed with a FE which in this casecomprises a nuclear localization signal (NLS). Thus, the presenttransport entity is particularly suitable for use in any method aimed attransfecting eucaryotic cells and directing nucleic acids of interestinto the nucleus of the cell. However, depending on the particular BEs,FEs and nucleic acids chosen in each case, the present transport entitymay be designed to enable an effective and specific transformation ofany nucleus with any gene or genetic element.

Thus, more specifically, an advantageous embodiment of the second aspectis a PNA-NLS complex according to the invention, which is described bythe general formula

. . . BE-L-FE . . .   (I),

wherein

the BE is a sequence of one or more PNA bases;

L denotes a linker sequence; and

the FE is an NLS sequence.

Said PNA-NLS complex is then hybridised to a plasmid containing a PNAtarget and a nucleic acid of interest, for example, a gene encoding apeptide, a protein, or an RNA. Said RNA may e.g. be a ribozyme, i.e. anRNA with enzymatic functions. In a specific embodiment, the NLS sequenceis a SV40 large T antigen protein or a fragment thereof, which exhibitsthe desired nuclear localizing signal properties. However, as theskilled in this field will realize, the choice of a suitable NLSsequence will depend on the intended future use. Thus, the present NLSmay be of any other origin or composition, as long as it fulfills thedesired functions of transferring a transport entity according to theinvention across the membrane and into the nucleus of the host cell.

In one specific and illustrating embodiment, the PNA-NLS complexaccording to the invention is described by formula III:

GCG CTC GGC CCT TTC (SEQ ID NO:1) L Pro Lys Lys Lys Arg Lys Val (SEQ IDNO:2) (III),

wherein L is described by formula IV:

NHCH₂CH₂OCH₂CH₂OCH₂CO₂H  (IV).

However, as the man skilled in the art will easily realise, variationsmay be made to these sequences while still providing an advantageoussynthetic transport entity within the scope of the present invention asdefined by the appended claims. Using the present invention, it ispossible to mimic the different functions of viruses and microorganismsby attaching functions directly to a nucleic acid or any otherbiological molecule and/or complex to be transferred to a cell. At thesame time, deleterious properties of native viral vectors are avoided bythe use of the present transfer entity.

Thus, the PNA comprising complex used as illustrating the invention willprovide excellent RNA and DNA hybridisation properties and biologicalstability and is easily produced by solid phase peptide synthesis (seee.g the international patent applications WO 95/01370 and WO 92/2072 andNielsen, P. E.: Peptide nucleic acid (PNA): “A lead for gene therapeuticdrugs”, Perspectives in Drug Discovery and Design, Vol. 4, pp. 76-84).

More specifically, in the construction of the transfer entity accordingto the invention, the present complex and carrier are attached to eachother by hybridisation of the BE of the complex to a BE target of thevector. Thus, in a particular embodiment, a PNA is hybridised to a PNAtarget sequence. Such a sequence specific hybridisation is easilyperformed by someone skilled in this field. (Hybridisation techniquesare for example generally described in “Nucleic Acid Hybridisation, APractical Approach”, Ed. Hames, B. D., and Higgins, B. D., IRL Press(1985); Gall and Pardue; Proc. Natl. Acad. Sci. USA 63: 378-383 (1969);and John et al., Nature 223:582-587 (1969)). Oligonucleotides may beprepared by any suitable method known to the skilled in this field, e.g.by direct chemical synthesis, such as the phosphotriester method ofNarang et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester methodof Brown et al., Meth. Enzymol. 68:109-151 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862 (1981); and the solid support method of U.S. Pat. No.4,458,066.

Different vectors and nucleic acids for use in construction of thecarriers are well known in the art and easily chosen by someone skilled.(For a general reference to laboratory procedures that may be used, seee.g. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd)ed., vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989.)

In a specific embodiment of the transfer entity according to theinvention, the carrier will also include a marker or a label, such as afluorescent label, etc., to enable detection and identification of thecells that have included the entity. In case of a plasmid carrier, thelabel may e.g. be a gene encoding a fluorescent protein, such as a greenfluorescent protein (GFP). In case of an oligonucleotide carrier, themarker is e.g. a fluorescent marker, such as Cy-3. Labelling and thedetection thereof are well known to the skilled in this field and aree.g disclosed in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,274,149; and 4,366,241.

Using the novel transfer entity according to the last mentionedembodiment of the invention, any nucleic acid sequences of interest,such as genes, may be introduced efficiently into a host cell due to theNLS ability to enter the nucleus. The transfection entity according tothe invention with its advantageous combined capability of efficient andspecific transfer of genetic elements is of great value in manydifferent applications, some of which will be disclosed in more detailbelow.

As illustrating the present invention, a method of transforming a cellmay be described by the following steps:

(i) providing a complex according to the invention by hybridising acarrier comprising at least one PNA target to the PNA domain of aPNA-NLS conjugate;

(ii) contacting the complex formed in step (a) with a cell to betransfected in the presence of a transfection reagent;

(iii) allowing the complex to enter the the nucleus of the cell; and

(iv) allowing genetic transformation to take place, wherein the PNAcomplex is used as a the nuclear translocation initiator. Thus, anytransforming sequences, which previously have been included in thevector on the requisite locations, may be transferred efficiently due tothe NLS ability to enter the nucleus of the cell and the PNA ability tospecifically bind to a PNA target on a vector which includes a nucleicacid sequence of interest.

As regards step (i), a variety of hybridisation formats is known tothose skilled in this field, such as sandwich assays and competition ordisplacement assays. Hybridisation techniques are for example generallydescribed in “Nucleic Acid Hybridisation, A Practical Approach”, Ed.Hames, B. D., and Higgins, B. D., IRL Press (1985); Gall and Pardue;Proc. Natl. Acad. Sci. USA 63: 378-383 (1969); and John et. al., Nature223:582-587 (1969).

Accordingly, firstly, the present method utilizes the transportcapability of the NLS, whereby it is brought into the nucleus of thehost cell together with any elements bound thereto. Once inside thenucleus, the NLS-protein complex may be dissolved and the proteinpartner to the FE (NLS) may exit the nucleus, while the transportedcarrier plasmid remains therein. Thus, secondly, the nucleic acid ofinterest present on the carrier may be utilised to perform a genetictransformation inside the target cell. Meanwhile, the protein partner tothe FE (NLS) is able to repeat its functions by accompanying additionalentities into the nucleus, thus contributing to the surprisingly hightransformation frequence obtained according to the present invention.

Thus, as mentioned above, Sebestyen et al. used a chemical coupling of anuclear localization signal to plasmids in methods of geneticmodification of cells, wherein no PNA is used. However, it appears thattheir method includes the serious drawback of also impairing the plasmidfunction. Thus, there are essential differences in methods betweenSebestyen et al. and the present invention, which may be a possibleexplanation to the substantial differences in transformation frequencyobtained.

Thus, in a specific embodiment, the transfection reagent is a polymertransfection reagent, such as PEI (polyethylene imine). PEI haspreviously been used in gene therapy experimental set-ups and has beenreported to be non-toxic in relevant dosages as well as capable ofproviding a high transfection efficiency. The pathway for PEItransfection is different from the standard lipid based transfectionreagents commonly used today. The polymer functions as a proton acceptorand is believed to disrupt the endosomes by osmotic stress, thusreleasing nucleic acids. In this context, it is to be noted that inother embodiments of the invention, using other specific functionalelements, such transfection agents as PEI, lipids etc, may be excluded.This depends on whether or not the functional elements included in thetransport entity are capable of providing a transport across the desiredmembrane. In a specific embodiment of the invention, the methodaccording to the invention further comprises an additional step (v),wherein the resulting transformation is confirmed by measuring apreviously included label or marker. The nature and identity of suchlabels and markers are also discussed elsewhere in this application.

In one advantageous embodiment of the above disclosed method accordingto the invention, the transformation defined in step (iv) introduces oneor more protein coding sequences by use of a plasmid vector. Thereby, anefficient transformation yielding a host cell expressing e.g. anexogenous, or non-native, protein or polypeptide is obtained. In analternative embodiment of the invention, the transformation according tostep (iv) may be used to introduce one or more gene regulatory sequencesin the host cell, whereby an otherwise silent gene may be expressed.This latter embodiment uses a technique known as gene activation, whichis described in detail e.g. in U.S. Pat. No. 5,641,670. In either one ofthe two above disclosed embodiments of the present method, the resultingtransformed cell may be used to produce substances such as proteinsuseful as medicaments.

In a further embodiment of the present method, the transformationdefined in step (iv) is aimed at repairing a mutation in the host cell,which e.g. may be obtained by base specific DNA repair. In analternative embodiment, said transformation is aimed at introducing amutation in a host cell. This may be desired, e.g. if the expression ofa gene is not desired due to the nature of the expression product or inthe production of animal models for the study of various geneticdiseases.

A further advantage of the present invention is that the illustratingPNA-NLS complex has a broad applicability and therefore the presentinvention may be used to transport more than 90% of all the plasmidsconventionally used on an everyday basis in research laboratoriesworldwide.

Accordingly, another aspect of the present invention is a recombinantcell produced by a method as disclosed above. The invention also relatesto animal models, such as mice, produced by a method according to theinvention specifically designed for the study of certain genomicdefects. General cloning techniques and methods of culturing cells arewell known to someone skilled in this field. (See e.g. Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold SprigHarbor Laboratory, Cold Spring Harbor, N.Y., 1989; Freshney: Culture ofAnimal Cells, A Manual of Basic Technique, 3^(nd) ed., Wiley-Liss, NewYork, N.Y. (1994)).

One particularly advantageous aspect of the present invention is the useof the PNA-NLS complex and the transfection method disclosed above ingene therapy. For such an application, the conjugate may be hybridisedto an oligonucleotide vector, such as a chimeric construct of DNA-RNA,PNA-DNA or any other combination. Gene therapy procedures have been usedto correct acquired and inherited genetic defects in a number ofcontexts. The ability to express artificial genes in humans, or animals,such as mammals, facilitates the prevention and/or cure of manyimportant diseases, often not amenable to treatment with othertherapies. However, presently available approaches to gene therapy makeuse of infectious vectors, such as retroviral vectors, which include thegenetic material to be expressed. Such approaches have limitations, suchas the potential of generating replication-competent virus during vectorproduction; recombination between the therapeutic virus and endogenousretroviral elements, potentially generating infectious agents with novelcell specificities; host ranges, or increased virulence andcytotoxicity; independent integration into large numbers of cells,increasing the risk of tumorigenic insertional event; limited cloningcapacity in the retrovirus (which restricts therapeutic applicability)and short-lived in vivo expression of the product of interest. Thus, theuse according to the present invention, wherein the PNA-NLS conjugatesor complexes according to the invention are used, avoids the limitationsand risks associated with the virus methods of the prior art. Forexample, previously, in the context of cystic fibrosis, adenovirusvectors have been used as a vector in gene therapy. Such a vector maygive rise to undesired and immunological responses, which accordinglywill be avoided by the advantageous use of the novel PNA-NLS conjugateaccording to the invention.

Consequently, the invention also relates to gene therapy methods assuch, wherein conjugates or complexes according to the invention areused, in certain applications together with at least one transfectionreagent, such as the above described polyethylene imine (PEI). Suchmethods are often aimed at repairing a mutated or defect gene, but mayalso be utilized to introduce a mutation, e.g. to prevent the expressionof an undesired protein or to produce an animal model for the study of acertain defect. One example of a disease that may be treated by genetherapy is cystic fibrosis, CF, which afflicts a large number ofpatients. CF may be amenable to plasmid mediated gene transfer, as thetarget organ is the lung which is fairly accessible. However, as thenumber of diseases for which a genetic defect is identified steadilyincreases, it is predicted that in the future, a large number ofadditional gene related conditions or sicknesses will be identified ashighly suitable for treatment by gene therapy according to the presentinvention. (For a general review of gene therapy methods, see e.g.Anderson, Science (1992) 256:808-813; Nabel and Felgner (1993) TIBTECH11: 211-217; Mitani and Caskey (1993) TIBTECH 11:162-166; Mulligan(1993) Science 926-932; Dillon (1993) TIBTECH 11:167-175; Miller (1992)Nature 357:455-460; Van Brundt (1988) Biotechnology 6(10):1149-1154;Vigne (1995) Restorative Neurology and Neuroscience 8: 35-36; Kremer andPerricaudet (1995) British Medical Bulletin 51(1) 31-44; Haddada et al.(1995) in Current Topics in Microbiology and Immunology; and Yu et al.,Gene Therapy (1994) 1:13-26.

A similar aspect of the present invention is the use of a transportentity or complex according to the invention to genetically modify cellsto be used in cell therapy. (For a disclosure of the fundamentals ofcell therapy methods, see e.g. Gage, F. H., Nature, vol. 392, Apr. 30,1998.) Consequently, the invention also relates to such cell therapymethods as well as to cells used therein that have been geneticallymodified by a method according to the present invention.

Accordingly, in a last aspect, the present invention also encompassesmethods of treatment, wherein an effective dose of the present transportentity is administered to a subject in need of treatment by genetherapy. Said treatment may be preventive and/or therapeutic, and it maybe directed to any disease or condition. Such conditions include variousgenetic defects, but are not limited thereto. Rather, further conditionsmay also be contemplated, wherein it is desired to accomplish a changeof the genetic environment, e.g. by insertion of a plasmid containing agene encoding a desired therapeutical function. Details regarding such atreatment scheme will be determined by the attending physician in eachcase depending on such factors as the condition to be treated, age andweight of the patient etc. Pharmaceutical preparations suitable for usein such methods are also within the scope of the invention.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing the target site in theanti-sense Cy-5 oligonucleotide hydridising to a sense PNA-NLS dualfunction peptide according to the invention. The PNA-NLS/oligonucleotidecomplex binds to the karyopherine α/β proteins. The complex is thentransported into the nucleus. Similarly the PNA target site was clonedinto the EGFP plasmid allowing nuclear transport subsequent tp PNA-NLShybridisation. Abbreviations: AS, anti-sense; S, sense; SV40 NLS, simianvirus 40 nuclear localization signal; and PNA, peptide nucleic acid.

FIG. 2(a) illustrates nuclear translocation of fluorescence labelledoligonucleotides. Arrows denote nuclei where the Cy-5 oligo is enriched.FIG. 2(b) illustrates Cy-5 channel showing the location of Cy-5AS-oligonuceotide. FIG. 2(c) illustrates Cy-3 channel showing thelocation of Cy-3 S-oligonucleotide.

FIG. 3 shows nuclear, cytoplasmic and Golgi-like ratio of Cy-5/Cy-3fluorescence. (a) Anti-sense oligo, nucleus; (b) Anti-sense oligo,Golgi-like; (c) Anti-sense oligo, cytoplasm; (d) Sense oligo, nucleus(e) Sense oligo, Golgi-like; and (f) Sense oligo, cytoplasm.

FIG. 4 is a shift assay of antisense and sense oligonucleotides withPNA-NLS dual function peptide according to the invention. (a)oligonucleotide with a target sequence (AS) for the PNA; (b) as in (a),but with prolonged exposure to show the weaker shift of the 1:10hybridisation; (c) oligonucleotide without a target sequence (S) for thePNA. Oligonucleotide concentrations were 2.64 pmol in all lanes. PNA-NLSwas added to 5′-labelled oligonucleotides at different molar ratios.Lane 1, 1:10; lane 2, 1:1; lane 3, 10:1; lane 4, 100:1; lane 5, 1000:1;and lane 6, 0:1.

FIG. 5 illustrates the effect of a transport entity according to theinvention: The effect of PNA-NLS when hybridised to PNA targetcontaining EGFP plasmids and the effect of removing the excess PNA-NLSmolecules from the transfection mix are shown. (a) PNA target sitecontaining EGFP plasmid transfected without PNA-NLS; (b) PNA target sitecontaining EGFP plasmid hybridised to PNA-NLS in 1:100 ratio; (c) PNAtarget site containing EGFP plasmid hybridised to PNA-NLS in 1:100 ratioand with excess PNA-NLS removed before transfection.

FIG. 6 shows lacZ and EGFP transfections with or without the addition ofPNA-NLS. EGFP transfections were also studied following removal ofunbound PNA-NLS (purified).

EXPERIMENTAL

Below, the present invention, will be further disclosed by way ofexamples. It is to be understood that the examples are merelyillustrating the invention and are not to be construed as limiting thescope of the invention as defined by the appended claims. All referencesbelow and elsewhere of this applications are hereby included herein byreference.

Materials and Methods

Cell Lines and Medium

COS-7, 3T3 and HeLa cells were used for transfections and cultivated inDMEM, 4500 mg/l glucose, 10 mM L-glutamine, 10% fetal calf serum and 50μg/ml gentamicine.

PNA-NLS

The peptide nucleic acid (PNA) was synthesised at PerspectiveBioSynthesis Ltd. The sequence of the PNA was chosen with the criteriaof being excluded from the plasmids as well as from known eucaryotic DNAsequences to avoid possible non-specific binding. The PNA peptides wereattached with the hydrophobic spacer Fmoc-NC₆O₃H₁₁—OH (Fmoc-AEEA-OH) toa stretch of amino acid residues, PKKKRKV (SEQ ID NO:2), the SV40 coreNLS. The complete sequence is GCGCTCGGCCCTTCC (SEQ IDNO:3)-linker-PKKKRKV (SEQ ID NO:2). Like peptides, PNA is synthesized ona polyethylene glycol-polystyren (PEG-PS) support with a peptide amidelinker, the linker yielding a PNA amide upon cleavage of the finalproduct (http://www.pbio.com/cat/synth/pna/pnacycle.htm).

Fluorochrome Labelled Oligonucleotides

Two fluorochrome labelled oligonucleotides were synthesised at CybergeneAB, AS-Cy-5 labelled, antisense to the PNA-NLS dual-function peptide,and S-Cy-3 labelled, sense to the PNA-NLS dual-function peptide. Theoligonucleotides were HPLC purified. The Cy-3 and Cy-5 fluorochromesubunits for linking to the oligonucleotides were purchased fromPerkin-Elmer.

Mobility Shift DNA-binding Assay Using Low Ionic Strength PAGE

Antisense- and sense-oligonuclecotides were end-labelled withT4-polynucleotide kinase using γ-³²P-ATP, >5000 mCi/mmol. The labelledoligonucleotides were incubated at room temperature with varying amountsof PNA-NLS. For separation of oligonucleotide/PNA-NLS complexes, 15%,low ionic-strength, non-denaturing polyacrylamide gel was used(“Mobility shift assay using low-ionic-strength PAGE”, Short protocolsin molecular biology, ed by Frederic M. Ausbel. Roger Brent, Robert E.Kingston, David D. Moore, J. G. Seidman, John A. Smith, Kevin Struhl,2^(nd) ed, 1992).

Transfections with PNA-NLS and Fluorochrome Labelled Oligonucleotides

Transfections were made with 25 kDa PEI as follows. PNA-NLS:AS:S weremixed at a molar ratio of 1:1:1 and heated to 90° C. The mix was allowedto slowly cool to room temperature to obtain conditions for optimalhybridisation of PNA-NLS to the AS-oligo. The mix was diluted with waterto a concentration of 0,05 μg/μl. To the mix of oligonucleotides andPNA-NLS, 1.44 μl of 0.1 M 25 kDa PEI solution was added per 2 μgnucleotide. The transfection solution was allowed to form complexes atroom temperature for 10 minutes and was subsequently mixed with 1 mlDMEM with 10% bovine serum and 100 μg/ml gentamicin. Themedium/transfection-complex mix was then added to the cells. Fortransfection, 10⁵ COS-7 cells were plated in 2 ml medium per well in6-well plates 8 h prior to the transfection to allow for cellattachment. Incubation time for oligonucleotide transfections was 10 h.All incubations were made at 37° C. in 5% CO₂.

Transfections With PNA-NLS, EGFP and lacZ Plasmids

The plasmid EGFP-N3 (Enhanced Green Fluorescent Protein (Clonetech)) wasmodified to include the target sequence for the PNA-NLS hybrid. TheEGFP-N3 plasmid was digested with AflII and ligated with aoligonucleotide fragment containing the PNA target sequence flanked byAflII sites. The fragment was cloned into a position outside anysequences known to be essential for EGFP gene function or plasmidfunction. Different clones of the construct were isolated containingdifferent numbers of PNA target sites. Expression of the EGFP and lacZgenes gave information about the status of the plasmid in respect tofunctionality of the reporter gene as measured by directly scoring thenumber of positive cells. Transfections were made with 25 kDa PEI asfollows. PNA-NLS:plasmid preparations were mixed at a molar ratio of100:1 and heated to 90° C. The mix was slowly cooled to room temperatureto allow for optimal hybridisation of PNA-NLS according to the inventionto the plasmid target site. The mix was diluted to a concentration of0.05 μg/μl. To the mix, 1.44 μl of 0.1 M 25 kDa PEI solution was addedper 2 μg of plasmid. The transfection solution was allowed to formcomplexes in room temperature for 10 minutes and was subsequently mixedwith 1 ml DMEM supplemented with 10% bovine serum and 100 μg/mlgentamicin. The transfection mix was then added to the target cells. Theprocedure for the lacZ plasmid was as above. For transfection, 10⁵ COS-7cells were plated in 2 ml medium per well in 6-well plates. This wasdone 8 h prior to the transfection to allow for cell attachment.Incubation time was 48 h to allow for maximum gene expression. Allincubations were made at 37° C. in 5% CO₂.

LacZ Staining

Transfected cells were fixed with 2% paraformaldehyde, 0.2%glutaraldehyde in 1×PBS. The staining solution, A, contained 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM magnesiumchloride. X-gal was dissoved in DMSO at a concentration of 40 mg/ml.Solution A was mixed with X-gal/DMSO solution at a 1:1 ratio, pre-warmedto 37° C. and added to the fixed cells. The cells were then incubatedover night at 37° C. and scored in a light microscope.

Fluorescence Microscopy and Image Analysis

Fluorescence microscopy was performed on live cells in a Leica DMRXAmicroscope with a cooled frame CCD (Charge Coupled Device) camera. Thesubsequent image analysis was performed with the software Slidebook2.1.4 from Intelligent Imaging Innovations Ltd. The increased nuclearuptake was calculated by masking the nucleus and then measure of thefluorescence from the Cy-5 (FIG. 2b) and the Cy-3 (FIG. 2c) spectra,respectively, and subtracting for the background fluorescence.

Results:

Nuclear Translocation of PNA-NLS Hybridised Oligonucleotides

In a first set of experiments, Cy-3 and Cy-5 fluorochrome labelled15-mer oligonucleotides were hydridised to the PNA-NLS at a molar ratioof 1:1. The principle of this technique is schematically outlined inFIG. 1. After PEI mediated transfection an increased nucleartranslocation of the Cy-5 fluorochrome labelled oligonucleotide/PNA-NLScomplex by 200-800% (FIGS. 2,3) was observed as compared to the controlCy-3 fluorochrome labelled oligonucleotide. When analysed, the cellscontained different levels of fluorescence in different cellularcompartments. The level of fluorescence was similar in the nuclei of allpositive cells. The cytoplasmic fluorescence varied from low to veryintense. To verify that the PNA-NLS binds to the cognate sequence in theoligonucleotide, radiactivelly labelled oligonucleotides were incubatedwith PNA-NLS and separated on low-ion strength, non-denaturingpolyacrylamide gels. As can be seen in FIG. 4, PNA-NLS binding wasspecific, as a shift was noticed using a molar ratio of 1:10 of PNA-NLSand the corresponding oligonucleotide, whereas a ratio of 10⁴:1 did notinfluence the oligo lacking the target site.

Nuclear Translocation of PNA-NLS Hybridised Plasmids

The PNA-NLS molecules were hybridised to PNA target sequence containingplasmids. LacZ or EGFP reporter constructs were used. The efficiency ofgene transfer was measured as frequency of EGFP expressing cells ornumber of blue cells after staining for lacZ activity. PEI mediatedtransfection of a lacZ or EGFP plasmids was enhanced 3-8-fold by theaddition of a 100:1 ratio of PNA-NLS peptide (FIGS. 5,6). The plasmidsboth contained 11 concatemeric PNA target sites cloned into a regionwithout known function (located 3′ of the poly-adenylation signal of thereporter gene). Initially dose-response analysis was carried outindicating that a 100:1 ratio of PNA-NLS:plasmid was optimal (data notshown). A lacZ plasmid containing 2 concatemeric PNA target sites didnot differ from wild-type efficacy (data not shown) and was not furtherstudied. The transfection efficacy was enhanced (8 fold as compared tocontrol) when the plasmid-PNA-NLS complex was purified from free PNA-NLSas shown in the case of the EGFP plasmid (FIGS. 5,6). This indicatesthat free PNA-NLS blocked the nuclear transport of the plasmid/PNA-NLScomplex thus impairing the nuclear translocation. When mixing a controlplasmid with PNA-NLS, no effect could be seen on transfection efficacy.The plasmid/PNA-NLS interaction does not seem to disturb the normalfunctions of a marker gene as shown by the expression of the reportergene in the complexes containing either lacZ or EGFP plasmids. Moreover,addition of 100-fold more free NLS peptide (ratio 10⁴:1 of NLS:plasmid)markedly reduced the transfection efficacy of plasmids containing thePNA site (not shown).

Discussion

The present invention demonstrates that a PNA molecule linked to an SV40NLS peptide can work as a nuclear targeting signal when hybridised to afluorescent labelled oligonucleotide or to a plasmid containing areporter gene. Similar results were obtained using DOTAP or 25 kD PEI astransfection reagents in HeLa, NIH-3T3 or COS-7 cells, demonstrating theversatility of the technique (data not shown). The method according tothe invention is of potential value for transfections in general and mayalso be applied in the context of gene therapy or DNA-vaccination. Theincreased uptake of nucleic acids into target cells may be vital forgene expression, as well as for the delivery of anti-sense constructs ormutation-inducing oligonucleotides. In the context of anti-senseactivity it should also be possible to apply a PNA-NLS construct alone.According to the present invention, a PNA target sequence,CGCGAGCCGGGAAGG (SEQ ID NO:4), was used, which does not exist in theunmodified EGFP or the lacZ plasmids that were studied. The interactionof PNA with its target sequence is highly specific and the PNA does notcross-hybridise to non-related sequences. The strong interaction betweenDNA and PNA also prevents the complex from dissociating (Knudsen H.,Nielsen P. E.: Antisense properties of duplex- and triplex-forming PNAs,Nucleic Acids Research 24(3) 494-500 (1996)).

While the present invention was in progress, a report appeared in whichdigitonin permeabilized HeLa cells were used to study nucleartranslocation after chemically linking the NLS peptide to a plasmid andinjecting it into the cytoplasm (Sebestyen M. G, Ludtke J. J., Bassik M.C., Zhang G., Budker V., Lukhtanov E. A., Hagstrom J. E., Wolff J. A.,DNA vector chemistry: The covalent attachment of signal peptides toplasmid DNA, Nature Biotechnology 16(1):80-5 (1998)). The resultsobtained by Sebestyen et al. also show increased nuclear import usingNLS linked plasmids. However, due to the chemical modifications inherentto this technique expression from reporter genes in the modifiedplasmids was blocked. In contrast, using PNA-NLS peptides expression wasmaintained. To this end, the location of PNA target sites in asupposedly non-vital region might be essential. For plasmids, it wasnoted that two PNA target sites had no effect on transfection efficacy,whereas 11 sites markedly increased efficacy. Even though the presentinventors did not study whether it is important to localize all targetsites to the same region in the plasmid or whether they could be spreadout evently, both alternatives are within the scope of protection asdefined by the appended claims. Moreover, extrapolating from the data ofSebestyen et al. (1998) it is proposed that a high local NLSconcentration is essential, as these authors saw no effect using ≧115NLS sequences per plasmid, whereas according to the present invention,an enhanced uptake was seen using a 250 bp concatemeric stretchharbouring 11 PNA-NLS target sites. However, it is not at presentcompletely clear whether or not PNA-NLS sequences bind to each targetsite, and accordingly, this will have to be tested by the skilled inthis field who uses the method according to the invention. The SV40 coreNLS was chosen of practical reasons, since it is one of the most studiedNLS sequences. SV40 linked proteins larger than 970 kDa have beenreported to translocate into the nucleus (Yoneda Y., Semba T., KanedaY., Noble R. L., Matsuoka Y., Kurihara T., Okada Y., Imamoto N.: “A longsynthetic peptide containing a nuclear localization signal and itsflanking sequences of SV40 T-antigen directs the transport of 1 gM intothe nucleus efficiently”, Experimental Cell Research 201(2):313-20(1992)). This should be compared with the molecular weight of the PNAtarget sequence containing EGFP plasmid of 2800 kDa. Furthermore, Zantaet al (Zanta M. A., Belguise-Valladier P., Behr J., Gene delivery: “Asingle nuclear localization signal peptide is sufficient to carry DNA tothe cell nucleus”, Proceedings of the National Academy of Sciences ofthe United States of America, 1999 96:91-96) disclosed, after theearliest priority date of the present patent application, an interestingpaper wherein ligation of an NLS-DNA hybrid to a purified reporter genefragment is used (8). The authors clearly demonstrate the feasibility ofa NLS approach. However, the method used by Zanta et al. (supra) is morecumbersome than the present method, since it involves restriction enzymecleavage of the plasmid and subsequent ligation of the NLS carryingoligonucleotide to the reporter gene fragment.

The present method and transport entity, such as the PNA-NLS system,will be efficient in assays and therapies involving transienttransfections as well as in systems where repair constructs are shuttledinto the nucleus for mutation repair. The system for nucleartranslocation based on the SV40 core NLS seemed to be saturated as aplateau of nuclear fluorescence was reached using fluorochrome labelledoligonucleotides. This is indicated by the fact that differentcytoplasmic levels of oligonucleotides still gave rise to similarnuclear levels of the targeted oligonucleotide, while the non-targetedoligonucleotide shows a relatively broad variation (FIG. 3b). Scoring ofnuclear translocation of oligonucleotides with Cy-5 labelled anti-senseoligonucleotide allowed for objectivity because of the infrared emissionof the Cy-5 fluorophore. The binding partner of SV40 NLS iskaryopherin-α which subsequently forms a complex with karyopherin-β(Miyamoto Y., Imamoto N., Sekimoto T., Tachibana T., Seki T., Tada S.,Enomoto T., Yoneda Y., Differential modes of nuclear localization signal(NLS) recognition by three distinct classes of NLS receptors, Journal ofBiological Chemistry, 272(42):26375-81 (1997)). The total amount of NLSis of importance, and will be determined by the skilled in this fieldutilizing the present method, as this transport mechanism can besaturated (Michaud N., Goldfarb D. S., Most nuclear proteins areimported by a single pathway, Experimental Cell Research, 208(1):128-36(1993)). Saturation of a nuclear import system is also the likelyexplanation for the findings according to the present invention thataddition of free NLS impairs nuclear transport. To further enhance thenuclear import of oligonucleotides, a mixture of PNA-NLS peptidescontaining a set of NLS sequences targeting different NLS pathways couldbe employed (Michaud et al, supra). This will ensure that saturation ofone pathway will not limit the nuclear translocation of the transfectedbio-molecules. To this end at least three different NLS receptorfamilies have been reported as exemplified by: Qipl, Rch 1 and NPI-1(Miyamoto el al., supra). According to the present invention, it issuggested that a way to mimic the lentivirus nuclear entry would be tobind the HIV-1 Matrix Association (MA) protein to the nucleic acidconstruct via a PNA-NLS peptide. The complex would then bind Vpr andmight subsequently be processed as a pre-integration complex (BukrinskyM. I., Haffar O. K., HIV-1 nuclear import: Matrix protein is back oncenter stage, this time together with Vpr. Molecular Medicine,4(3):138-43 (1998)). The function of the MA-Vpr complex is proposed tobe analogous to the extended SV40 NLS sequence described by Xiao et al.(1997) (Xiao C. Y., Hubner S., Jans D. A., SV40 large tumor antigennuclear import is regulated by the double-stranded DNA-dependent proteinkinase site (serine 120) flanking the nuclear localization sequence,Journal of Biological Chemistry, 272(35):22191-8 (1997)). In the priorart, proteins enhancing various aspects of nucleic acid delivery havebeen studied (Boussif O., Lezoualch F., Zanta M. A., Mergny M. D.,Scherman D., Demeneix B., Behr J. P., A versatile factor for gene andoligonucleotide transfer into cells in culture and in vivo:polyethylenimine, Proceedings of the National Academy of Sciences of theUnited States of America 92(16):7297-301 (1995)). According to thepresent invention, it is suggested that such functions, in addition tothe NLS-mediated transfer, could be directly targeted to the nucleicacid using the PNA approach.

4 1 15 DNA Artificial Sequence Peptide Nucleic Acid linked to SEQ ID NO21 gcgctcggcc ctttc 15 2 7 PRT Artificial Sequence Nuclear LocalizaitonSignals linked to SEQ ID NO1 2 Pro Lys Lys Lys Arg Lys Val 1 5 3 14 DNAArtificial Sequence Peptide Nucleic Acid linked to SEQ ID NO2 3gcgctcggcc cttc 14 4 15 DNA Artificial Sequence Peptide Nucleic Acidtarget sequence 4 cgcgagccgg gaagg 15

What is claimed is:
 1. A synthetic transport complex for transferring anucleic acid of interest across a biological membrane into a cell,wherein the complex is comprised of two or more functional elements(FE), each of which is complexed to a binding element (BE) in the formof a peptide nucleic acid (PNA), and a nucleic acid carrier, whichcomprises at least two BE target sequences and a nucleic acid ofinterest in a vector; said carrier being hybridized to said complexusing the BE-BE interaction.
 2. The transport complex according to claim1, wherein said two or more FEs provide different functions.
 3. Thetransport complex according to claim 1, wherein said vector is a plasmidor an oligonucleotide.
 4. The transport complex according to claim 1,wherein the carrier includes a detectable marker element.
 5. Thetransport complex according to claim 1, wherein the nucleic acid ofinterest is a gene encoding a peptide, a protein or an RNA.
 6. Thetransport complex according to claim 1, wherein said BE and FEs areseparated by linker elements.
 7. The transport complex according toclaim 1, which comprises more than one FE-BE-complex, each one of whichis hybridized to a separate BE target sequence present on the samecarrier.
 8. The transport complex according to claim 1, wherein the FEis a nuclear localization signal (NLS), or a fragment thereof exhibitingnuclear localizing signal properties.
 9. The transport complex accordingto claim 1, wherein the FE is a protein exhibiting properties enablingboth membrane translocation and nuclear transport.
 10. A method fortransferring a nucleic acid of interest across a biological membrane ofa target cell comprising administering to the cell the synthetictransport complex according to claim
 1. 11. The method according toclaim 10, wherein in said transport complex said two or more FEs providedifferent functions.
 12. The method according to claim 10, wherein insaid transport complex said BE and FEs are separated by linkerelement(s).
 13. The method according to claim 10, wherein in saidtransport complex the carrier provided is a plasmid or anoligonucleotide vector comprising said nucleic acid of interest and atleast one target sequence.
 14. The method according to claim 10, whereinin said transport complex a detectable marker element is inserted insaid carrier.
 15. The method according to claim 10, wherein in saidtransport complex the nucleic acid of interest is a gene encoding apeptide, a protein or an RNA.
 16. The method according to claim 10,wherein said complex comprises more than one FE-BE complex, each one ofwhich is hybridized to a separate BE target sequence present on the samecarrier.
 17. The method according to claim 10, wherein the biologicalmembrane is a cell wall.
 18. The method according to claim 10, whereinthe biological membrane is a nuclear membrane, and wherein at least onefunctional element (FE) of said two or more functional elements is aprotein, which enables both membrane translocation and nuclear transportof the nucleic acid of interest.
 19. The method according to claim 10,wherein in said transport complex the FE is a nuclear localizationsignal (NLS), or a fragment thereof exhibiting nuclear localizing signalproperties.
 20. The method according to claim 10, wherein in saidtransport complex the FE is a protein provided in said complex, whichenables both membrane translocation and nuclear transport of the nucleicacid of interest.
 21. A kit comprising components for making a transportentity capable of transferring a nucleic acid of interest across abiological membrane into a cell, which kit comprises at least twobinding elements (BE) in the form of a peptide nucleic acid (PNA); twoor more functional elements (FE); a plasmid containing said nucleic acidof interest; an oligonucleotide comprising a target for each of said BEsand being suitable for cloning in said plasmid; and optionally reagentssuitable for such transfer.
 22. The kit according to claim 21, whereinsaid two or more FEs provide different functions.
 23. The kit accordingto claim 21, wherein at least one functional element (FE) is a nuclearlocalization signal (NLS), or a fragment thereof exhibiting nuclearlocalizing signal properties.
 24. The kit according to claim 21, whereinthe FE is a protein provided in said complex, which enables bothmembrane translocation and nuclear transport of the nucleic acid ofinterest.
 25. The transport complex according to claim 8, wherein saidNLS is a SV40 large T antigen protein.
 26. The transport complexaccording to claim 9, wherein the FE is an HIV protein.
 27. Thetransport complex according to claim 26, wherein said HIV protein isTAT.
 28. The method according to claim 19, wherein in said transportcomplex said NLS is a SV40 large T antigen protein.
 29. The methodaccording to claim 20, wherein in said transport complex the FE is anHIV protein.
 30. The method according to claim 29, wherein said HIVprotein is TAT.
 31. The kit according to claim 23, wherein said NLS is aSV40 large T antigen protein.